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1.
Article in Chinese | WPRIM | ID: wpr-232198

ABSTRACT

<p><b>OBJECTIVE</b>To determine the karyotype of a boy suspected to have Cri du Chat syndrome with severe clinical manifestations, and to assess the recurrence risk for his family.</p><p><b>METHODS</b>High-resolution GTG banding was performed to analyze the patient and his parents. Fluorescence in situ hybridization (FISH) with Cri du Chat syndrome region probe as well as subregional probes mapped to 5pter, 5qter, 18pter, 18qter, and whole chromosome painting probe 18 was performed to analyze the patient and his parents. In addition, single nucleotide polymorphism-based arrays (SNP-Array) analysis with Affymetrix GeneChip Genome-wide Human SNP Nsp/Sty 6.0 were also performed to analyze the patient.</p><p><b>RESULTS</b>Karyotype analysis indicated that the patient has carried a terminal deletion in 5p. FISH with Cri du Chat syndrome region probe confirmed that D5S23 and D5S721 loci are deleted. SNP-Array has detected a 15 Mb deletion at 5p and a 2 Mb duplication at 18p. FISH with 5p subtelomeric probes and 18p subtelomeric probe further confirmed that the derivative chromosome 5 has derived from a translocation between 5p and 18p, which has given rise to a 46,XY,der(5)t(5;18)(p15.1;p11.31)dn karyotype.</p><p><b>CONCLUSION</b>A de novo 5p partial deletion in conjunction with a cryptic 18p duplication has been detected in a boy featuring Cri-du-Chat syndrome. His parents, both with negative findings, have a low recurrence risk. For its ability to detect chromosomal imbalance, SNP-Array has a great value for counseling of similar patients and assessment of recurrence risks.</p>


Subject(s)
Child, Preschool , Chromosome Banding , Chromosome Deletion , Chromosomes, Human, Pair 18 , Chromosomes, Human, Pair 5 , Cri-du-Chat Syndrome , Diagnosis , Genetics , Humans , In Situ Hybridization, Fluorescence , Male , Phenotype , Polymorphism, Single Nucleotide , Trisomy
2.
Article in Chinese | WPRIM | ID: wpr-237243

ABSTRACT

<p><b>OBJECTIVE</b>To screen for potential mutations of KIT gene for two Chinese families affected with piebaldism in order to facilitate genetic counseling and assisted reproduction.</p><p><b>METHODS</b>Peripheral blood samples were collected from 2 patients of family 1 and the proband and 3 unaffected members of family 2 for the extraction of DNA and RNA. PCR-sequencing and reverse transcription PCR-sequencing were used to screen KIT mutations.</p><p><b>RESULTS</b>All of the patients from family 1 were found to carry heterozygous IVS12+2-+7delinsACATCTTTA, a splicing mutation undocumented in the human gene mutation data base (HGMD) database. This mutation has resulted in c.1765-1779del in cDNA and p.Gly592Ala/del:E12, which has led to skipping of exon 12 and no expression of cDNA. The proband from family 2 has carried a heterozygous c.2401A>C mutation in KIT gene. The same mutation was not found in unaffected members.</p><p><b>CONCLUSION</b>We have attained definite diagnosis for both families, which has facilitated genetic counseling and assisted reproduction for our patients and their family members.</p>


Subject(s)
Adult , Asians , Genetics , Base Sequence , Child , China , Female , Frameshift Mutation , Humans , Male , Molecular Sequence Data , Pedigree , Piebaldism , Genetics , Point Mutation , Proto-Oncogene Proteins c-kit , Genetics , Young Adult
3.
Article in Chinese | WPRIM | ID: wpr-267683

ABSTRACT

<p><b>OBJECTIVE</b>To isolate human amniotic mesenchymal cells (hAMCs) and investigate their transdifferentiation ability into islet-like cells in vitro.</p><p><b>METHODS</b>Human amnion was treated with the trypsin/EDTA to remove the amniotic epithelial cells and then incubated with collagenase I and dispase at 37 degrees celsius; overnight. The cells were collected by centrifugation and identified for the expressions of vimentin and SSEA-4 using immunofluorescence assay and for CD29, CD90, CD34, and CD45 using flow cytometry. RT-PCR was performed to detect the expressions of ACTG2, ACTA2, MMP2, Cripto, Sox2, LEFTYA, nanog, and Oct-4 in the cells. The differentiation potential of the isolated cells into inslet-like cells was assessed after a 14-day induction with the inducing factors by RT-PCR and immunofluorescence assay.</p><p><b>RESULTS</b>The hAMCs were capable of in vitro proliferation and passaging for 10 passages while retaining the normal karyotype. The isolated cells were positive for staining of vimentin and SSEA-4 and negative for CD34 and CD45; the CD29 and CD90 cells accounted for (91.5∓9.93)% and (48.7∓9.47)% of the cells, respectively. The hAMCs expressed several pluripotency-related genes, including Cripto, Sox2, LEFTYA, nanog, and Oct-4. After induction, endocrine-related genes were expressed in the islet-like cells, including PDX1, ngn3, insulin and glucagon.</p><p><b>CONCLUSION</b>We have successfully established the method for isolating hAMCs, which possess the potential of differentiation into islet-like cells in vitro.</p>


Subject(s)
Amnion , Cell Biology , Cell Culture Techniques , Methods , Cell Transdifferentiation , Physiology , Cells, Cultured , Female , Humans , Islets of Langerhans , Cell Biology , Mesenchymal Stem Cells , Cell Biology
4.
National Journal of Andrology ; (12): 13-16, 2011.
Article in Chinese | WPRIM | ID: wpr-266220

ABSTRACT

<p><b>OBJECTIVE</b>To explore the significance of karyotype analysis in screening sperm donors.</p><p><b>METHODS</b>From January 1, 2004 to December 31, 2008, a total of 2537 potential sperm donors passed our preliminary screening, and all were routinely karyo-typed via peripheral blood. Follow-ups were conducted on the pregnancy outcome and congenital malformation after artificial insemination with the sperm from the qualified donors.</p><p><b>RESULTS</b>Among the 2537 qualified sperm donors, 2362 were of the normal karyotype 46, XY and 135 showed polymorphism. Abnormal karyotype was found in 6 cases, and controversial abnormal karyotype in 34.</p><p><b>CONCLUSION</b>Karyotype analysis can reduce the risk of chromosomal disease in neonates from artificial insemination, and genetic counseling for abnormal karyotype sperm donors may help them solve their future reproductive problems.</p>


Subject(s)
Adult , Chromosome Aberrations , Chromosome Disorders , Genetic Testing , Gonadal Dysgenesis, 46,XY , Genetics , Humans , Karyotyping , Male , Sperm Banks , Tissue Donors , Young Adult
5.
Article in Chinese | WPRIM | ID: wpr-326979

ABSTRACT

<p><b>OBJECTIVE</b>To identify the F VIII gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis.</p><p><b>METHODS</b>PCR, denaturinghigh performance liquid chromatogramphy (DHPLC) and DNA sequencing technologies were applied to screen the F VIII gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14(DXS 52), intron 13 (CA)n and EX18/Bcl I of the F VIII gene in the HA families. In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F VIII gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism.</p><p><b>RESULTS</b>Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism (SNP) were identified in 10 the HA families. Among them, c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT, c.4880_4881insA and c.5000G to A were novel mutations or polymorphism. No missense mutations c.878A G, c.1015A to G and c.6870G to T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined to in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient.</p><p><b>CONCLUSION</b>Six novel mutations, i.e., c.878A to G, c.1015A to G, c.6870G to T, c.1282delA, c.3072_3073insT and c.4880_4881insA, were identified in this study. PCR, DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.</p>


Subject(s)
Chromosomes, Human, X , DNA Mutational Analysis , Methods , DNA Restriction Enzymes , Genetics , Factor VIII , Genetics , Female , Genetic Testing , Methods , Hemophilia A , Diagnosis , Genetics , Heterozygote , Humans , Male , Mutation , Pedigree , Polymorphism, Single Nucleotide , Prenatal Diagnosis , Methods , Sequence Analysis, DNA , Methods
6.
Article in Chinese | WPRIM | ID: wpr-326953

ABSTRACT

<p><b>OBJECTIVE</b>Mutation screening was performed in a pedigree of Glanzmann's thrombasthenia (GT) and prenatal diagnosis was performed.</p><p><b>METHODS</b>In this study, reverse transcription-PCR-sequencing and PCR-sequencing, as well as restriction fragment length polymorphism(RFLP) and A/T-cloned-sequencing, were used to screen the ITGA2B and ITGB3 mutation in a pedigree with Glanzmann's thrombasthenia in the RNA and DNA level. Prenatal diagnosis was performed for this pedigree.</p><p><b>RESULTS</b>Deletion of 99 bps was found in the cDNA of the patient in the pedigree, leading to deletion of 33 codons (from codon 160 to 192). After genomic analysis, the patient was found to be a compound heterozygote of c.374C to G mutation and intron 4(IVS-4) + 5 G to C mutation. The two mutations were inherited from the parents. IVS-4 + 5 G to C mutation was a point mutation in the splice site, while c.374C to G mutation was out of the splice site. But both of them resulted in the same splice pattern in RNA. The two mutations were novel mutations which have not been reported in Human Gene Mutation Database (HGMD) and the mutation data base of Glanzmann's thrombasthenia. The results of ITGB3 gene screening is normal in the proband and his parents.</p><p><b>CONCLUSION</b>Two novel mutation, c.374C to G and IVS-4 + 5 G to C were found in this study, which might be the cause of GT in the pedigree.</p>


Subject(s)
Base Sequence , Child, Preschool , Female , Gene Order , Genetic Testing , Genotype , Humans , Male , Molecular Sequence Data , Mutation , Genetics , Pedigree , Platelet Glycoprotein GPIIb-IIIa Complex , Chemistry , Genetics , Pregnancy , Prenatal Diagnosis , Protein Conformation , Thrombasthenia , Diagnosis , Genetics
7.
Article in Chinese | WPRIM | ID: wpr-326932

ABSTRACT

<p><b>OBJECTIVE</b>To screen mutations of tuberous sclerosis complex (TSC) patients to confirm a clinical diagnosis of TSC, and to perform prenatal diagnosis for families with mutations.</p><p><b>METHODS</b>In this study, PCR-denaturing high-performance liquid chromatography(DHPLC), supplemented with sequencing when necessary, was used to screen TSC1 and TSC2 mutations in 21 patients from 19 pedigrees visited author's hospital in the last five years. For novel mutations, one hundred unrelated healthy individuals were screened to exclude the possibility of polymorphism.</p><p><b>RESULTS</b>Seventeen different mutations were found in 21 patients of 19 pedigrees with 13 being novel mutations, including c. 2672delA, c. 2672insA of TSC1 gene and c.4918insCGCC, c.1143delG, Intron27+1 G>A, c.1957-1958delAG, Intron5+1 G>A, c.910insCT, c.2753 C>G, c.4078dupAGCAAGTCCAGCTCCTC, Intron 11 -1 G>A, Intron 14+1 G>A, c.684 C>A of TSC2 gene, indicating a high frequency of de novo mutations in TSC. Three of these mutations were in the TSC1 gene (N762S, c.2672insA and c. 2672delA), while all remaining 14 were in the TSC2 gene. Prenatal diagnosis for TSC was performed for 7 fetuses from these pedigrees. The six fetuses that tested negative for TSC mutations were carried to term and, to date, none of these children has shown symptoms of TSC.</p><p><b>CONCLUSION</b>Author's data showed that a mutation detection rate of tuberous sclerosis was 89.5%(17/19) among patients in author's hospital. The ratio of TSC2 and TSC1 mutations was about 1:1 in the familial cases, but TSC2 mutation was more common than TSC1 mutation in sporadic cases. Author's data demonstrated that birth of TSC children for those with familial history of TSC could be prevented through prenatal diagnosis.</p>


Subject(s)
Base Sequence , DNA Mutational Analysis , Methods , Female , Humans , Male , Pedigree , Polymorphism, Single Nucleotide , Genetics , Pregnancy , Prenatal Diagnosis , Methods , Retrospective Studies , Tuberous Sclerosis , Diagnosis , Genetics
8.
Article in Chinese | WPRIM | ID: wpr-333881

ABSTRACT

<p><b>OBJECTIVE</b>To establish a method for isolating and culturing human amniotic epithelial cells (hAECs) in a serum-free medium and investigate their transdifferentiation ability into islet-like cells.</p><p><b>METHODS</b>The culture condition of hAECs was optimized using DMEM with different supplements. The genetic stability of the tenth-passage cells was assessed by chromosome analysis and G-banding method. The stem cell characteristics of the cells were identified by examination of the surface markers using immunofluorescence methods. The endocrine-related genes and hormones of the cells were tested after induced differentiation into islet-like cells.</p><p><b>RESULTS</b>The hAECs allow stable passaging in the presence of 10 ng epidermal growth factor (EGF) in the culture medium. After 10 passages, the cells maintained a normal karyotype and G-banding profile. The hAECs expressed many multi-potent stem cell markers, including SSEA4, TRA-1-60, and TRA-1-81. After induced differentiation, the endocrine-related genes were expressed in the islet-like cells, including PDX1, ngn3, insulin and glucagon. Insulin secretion increased in the differentiated islet-like cells in response to high glucose exposure.</p><p><b>CONCLUSION</b>We established a method for isolating and expanding the hAECs in a serum-free medium. hAECs possess stem cell characteristics and can be induced to differentiate into islet-like cells in vitro.</p>


Subject(s)
Amnion , Cell Biology , Cell Culture Techniques , Methods , Cell Differentiation , Cells, Cultured , Epithelial Cells , Cell Biology , Bodily Secretions , Humans , Insulin , Bodily Secretions , Islets of Langerhans , Cell Biology , Stem Cells , Cell Biology , Bodily Secretions
9.
Article in Chinese | WPRIM | ID: wpr-234399

ABSTRACT

<p><b>OBJECTIVE</b>To establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD).</p><p><b>METHODS</b>Twelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed.</p><p><b>RESULTS</b>The amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods.</p><p><b>CONCLUSION</b>PEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.</p>


Subject(s)
Comparative Genomic Hybridization , Methods , DNA Primers , Genetic Testing , Methods , Humans , Karyotyping , Methods , Nucleic Acid Amplification Techniques , Methods , Nucleic Acid Hybridization , Methods , Oligonucleotides , Chemistry , Preimplantation Diagnosis , Methods
10.
Article in Chinese | WPRIM | ID: wpr-234368

ABSTRACT

<p><b>OBJECTIVE</b>To determine the karyotype of a patient with Prader-Willi-like syndrome features.</p><p><b>METHODS</b>Chromosomal high resolution banding was carried out to analyze the karyotype of the patient, and methylation-specific PCR was used to analyze the imprinting region of chromosome 15. Subtelomeric region was screened by multiplex ligation-dependent probe amplification (MLPA), and fluorescent in situ hybridization (FISH) and real-time quantitative PCR were further performed to identify the deleted region.</p><p><b>RESULTS</b>No abnormality was discovered by high resolution karyotype analysis and methylation-specific PCR studies. MLPA analysis showed that the patient had a deletion of 1p subtelomeric area, which was confirmed by FISH analysis. The deleted region was shown within a 4.2 Mb in the distal 1p by 3 BAC FISH probes of 1p36 combined with real-time PCR technique. Family pedigree investigation showed the chromosome abnormality was de novo. Therefore, partial monosomy 1p36 was likely responsible for the mental retardation of the patient.</p><p><b>CONCLUSION</b>Molecular cytogenetic techniques should be performed to those patients with Prader-Willi-like syndrome features, to determine their karyotypes.</p>


Subject(s)
Child , Chromosome Deletion , Chromosomes, Human, Pair 1 , Genetics , Female , Humans , Karyotyping , Prader-Willi Syndrome , Genetics
11.
Article in Chinese | WPRIM | ID: wpr-330828

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the relationship between CTLA4 +49G/A SNP polymorphism and the susceptibility to hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>CTLA4+49G/A single nucleotide polymorphism (SNP) was analyzed by DNA sequencing in 165 control subjects, 155 patients with chronic hepatitis B (CHB) and 149 HCC patients. Serum HBsAg, HBeAg and AFP levels were measured in all the subjects.</p><p><b>RESULTS</b>In HCC and CHB groups, the genotype frequency was 40.3% and 50.0% for GG , and 59.7% and 50.0% for AG+AA, respectively, while the genotype frequency was 61.8% for GG and 38.2% for AG+AA in the control group. In HCC group, CHB group and controls, the A allele frequencies was 44.6%, 37.4% and 28.8%, and the G allele frequencies was 55.4%, 62.6% and 71.2%, respectively. Significant differences were found not only in the allele frequencies (P<0.05) but also in AA and combined (AA+AG) genotype frequencies (P<0.05) between the 3 groups.</p><p><b>CONCLUSION</b>+49G/A SNP of the CTLA4 gene can be associated with HBV and HBV-related HCC.</p>


Subject(s)
Adult , Asians , Genetics , CTLA-4 Antigen , Genetics , Carcinoma, Hepatocellular , Genetics , Virology , Case-Control Studies , Female , Gene Frequency , Genetic Predisposition to Disease , Genotype , Hepatitis B , Genetics , Hepatitis B Surface Antigens , Blood , Hepatitis B e Antigens , Blood , Hepatitis B virus , Humans , Liver Neoplasms , Genetics , Virology , Male , Middle Aged , Polymorphism, Single Nucleotide
12.
Article in Chinese | WPRIM | ID: wpr-330781

ABSTRACT

<p><b>OBJECTIVE</b>To construct pGEX-KG/mTSARG3 recombinant vector and prepare the fusion protein to obtain rabbit polyclonal antibody against mTSARG3.</p><p><b>METHODS</b>The open reading frame (ORF) of mTSARG3 gene was amplified from mouse testis cDNA library by PCR. The products were cloned into pGEM-T Easy vectors and sequenced. The recombinant plasmids were digested by EcoRI and SalI and subcloned into PGEX-KG vector. After identification by DNA sequence analysis, the recombinant plasmids were transformed into component E.coli BL21 cells, and the GST/mTSARG3 fusion protein was expressed with IPTG induction. The anti-mTSARG3 polyclonal antibody was produced by immunization of rabbits with the fusion protein. The resultant antibody was purified by antigen column and used for Western blotting for detecting mTSARG3 expression.</p><p><b>RESULTS</b>The recombinant vector pGEX-KG/mTSARG3 was successfully constructed. GST/mTSARG3 fusion protein was expressed abundantly at 4 h after IPTG induction and polyclonal antibodies were obtained. Western blot analysis demonstrated that the antibodies specifically detected mTSARG3 expression in E.coli BL21.</p><p><b>CONCLUSION</b>We have successfully constructed pGEX-KG/mTSARG3 recombinant vector and obtained rabbit polyclonal antibody, which may facilitate further investigation of the role of mTSARG3 in spermatogenesis.</p>


Subject(s)
Animals , Antibodies , Allergy and Immunology , Base Sequence , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Heat-Shock Proteins , Genetics , Allergy and Immunology , Male , Mice , Molecular Sequence Data , Rabbits , Recombinant Fusion Proteins , Genetics , Allergy and Immunology
13.
Article in Chinese | WPRIM | ID: wpr-233731

ABSTRACT

<p><b>OBJECTIVE</b>To characterize the time course of spontaneous differentiation of in vitro cultured human embryonic stem cells (hESCs) into hematopoietic cells to provide experimental evidence for induction of hematopoietic commitment of hESCs.</p><p><b>METHODS</b>In human embryoid bodies (hEBs) derived from spontaneous differentiation of chESC3, a hESC cell line we established previously, the expressions of such genes as KDR, Bmi1, Scl and gata2 were detected by RT-PCR every other day during the 12-day differentiation to monitor the process of the hematopoiesis. The hematopoietic stem cell marker CD34 was examined using flow cytometry to evaluate the efficiency of hematopoietic differentiation of the cells on days 6, 8, 10 and 12. The spontaneously differentiated hESCs were seeded in the hematopoietic colony culture system to study the hematopoietic colony forming ability. Immunocytochemical staining for CD45 was performed on the hEBs to examine the emergence of mature hematopoietic cells.</p><p><b>RESULTS</b>The expressions of the hematopoietic stem cell-related genes KDR and Bmi-1 were detected in the hESCs, and on days 4 to 6, the two genes were upregulated with prolonged cuture of the hEBs. Scl and gata2 gene expressions were detected since 6-8 days of culture and maintained high expressions till day 12. Flow cytometry revealed a gradual increase in CD34-positive cells in the culture, with positivity rates on days 6, 8, 10, and 12 of (1.4-/+0.4)%, (3.4-/+1.3)%, (5.5-/+2.2)%, and (5.1-/+1.7)%, respectively. The numbers of CD43-positive cell colonies on days 6, 8, 10, and 12 were 0, 7-/+2, 37-/+11, and 89-/+29 in each 10(5) cells, respectively. Immunocytochemical staining identified CD45-positive cells on days 10, 12, 15, and 18 in the cell colonies, with the positive cell numbers of 0, 40.5-/+15.09, 178.6-/+55.89, and 253.0-/+52.04, respectively.</p><p><b>CONCLUSION</b>The hESCs undergo spontaneous hematopoietic differentiation in 3 stages, including the differentiation into germ layer-specific cells (days 6-8), expansion period of the hematopoitic progenitors (days 8-12), and maturation of the hematopoietic cells (after day 15).</p>


Subject(s)
Animals , Antigens, CD34 , Metabolism , Basic Helix-Loop-Helix Transcription Factors , Genetics , Cell Culture Techniques , Cell Differentiation , Embryonic Stem Cells , Cell Biology , Metabolism , GATA2 Transcription Factor , Genetics , Gene Expression Regulation , Hematopoietic Stem Cells , Cell Biology , Metabolism , Humans , Mice , Nuclear Proteins , Genetics , Polycomb Repressive Complex 1 , Proto-Oncogene Proteins , Genetics , Repressor Proteins , Genetics , T-Cell Acute Lymphocytic Leukemia Protein 1 , Time Factors
14.
Article in Chinese | WPRIM | ID: wpr-282606

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the roles of human fetal liver stromal cells (hFLSCs) and human fetal bone marrow stromal cells (hFBMSCs) in the hematopoietic differentiation of human embryonic stem cells and analyze their gene expression profile changes.</p><p><b>METHODS</b>The embryonic bodies on day 4 were cocultured with hFLSCs or hFBMSC in the presence of cytokines. Flow cytometry was performed after 8 days of induction to detect the expressions of the hemangioblast markers KDR and CD34, and the differential gene expression profiles between hFBMSC and hFLSCs were examined by cDNA microarray analysis.</p><p><b>RESULTS</b>Eight days after the induction, (1.06-/+0.20)% of the hFLSCs and (8.8-/+1.49)% of the hFBMSCs were positive for KDR, with the positivity rates for CD34 of (1.25-/+0.16)% and (9.17-/+2.10)%, respectively. In hFLSCs and hFBMSCs cultures, 0.9-/+0.36 and 10.6-/+0.63 hemagioblast-like cell colonies were found, respectively. cDNA microarray analysis showed that 240 genes were highly expressed in hFBMSCs, and 21 genes related to secreted cytokines, cell adhesion molecules and extracellular matrix proteins were highly expressed.</p><p><b>CONCLUSION</b>The microenvironment including the cell matrix protein and cytokines secreted by the hFBMSCs might play an important role in hemangioblastic differentiation of human bone marrow stromal cells in vitro.</p>


Subject(s)
Antigens, CD34 , Genetics , Metabolism , Bone Marrow Cells , Cell Biology , Cell Differentiation , Physiology , Coculture Techniques , Embryonic Stem Cells , Cell Biology , Fetus , Gene Expression Profiling , Hematopoietic Stem Cells , Cell Biology , Humans , Liver , Cell Biology , Oligonucleotide Array Sequence Analysis , Stromal Cells , Physiology , Vascular Endothelial Growth Factor Receptor-2 , Genetics , Metabolism
15.
National Journal of Andrology ; (12): 974-979, 2009.
Article in Chinese | WPRIM | ID: wpr-252847

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the relationship of the mutation of the spermatogenesis-associated gene KLHL-10 with azoospermia, oligospermia and asthenospermia.</p><p><b>METHODS</b>Genomic DNA was extracted from the peripheral blood samples of 325 patients with idiopathic azoospermia (n = 11), oligozoospermia (n = 196) or asthenospermia (n = 118) and 100 fertile male controls. KLHL-10 mutations were detected for all the DNA specimens by PCR, DHPLC and sequencing techniques.</p><p><b>RESULTS</b>A novel heterozygous mutation (C88 --> A) was identified in exon 1 from 1 oligospermia patient and 3 fertile male controls and another one (C424 --> A) confirmed in exon 2 from 4 fertile controls, 3 oligospermia patients and 1 asthenospermia man. Both of the mutations were synonymous, but neither missense mutation nor microdeletion of the KLHL-10 gene was found.</p><p><b>CONCLUSION</b>The KLHL-10 gene is not a major contributor to azoospermia, oligospermia or asthenospermia in Chinese population. The value of this gene in the diagnosis of male infertility remains to be further investigated.</p>


Subject(s)
Adult , Asthenozoospermia , Genetics , Azoospermia , Genetics , Case-Control Studies , Exons , Gene Frequency , Genotype , Humans , Male , Mutation , Oligospermia , Genetics , Proteins , Genetics , Young Adult
16.
Article in Chinese | WPRIM | ID: wpr-253313

ABSTRACT

The aim of this study was to establish the method of isolating and culturing endothelial progenitor cells (EPCs) from human umbilical cord blood. Mononuclear cells (MNCs) from human umbilical cord blood were cultured by using culture system supplemented with endothelial cell-conditioned medium. The obtained two types of cells were purified by picking up colonies, identified by uptake of acetylated low-density lipoprotein (Ac-LDL) and binding to lectin [Ulex European Agglatinin (UEA-1)], and were analyzed for the expression of markers by flow cytometry. The results showed that there were significant differences between two types of cells in proliferation, so they were referred as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs), respectively. They were in accordance with the standards of EPCs, could uptake DiI-Ac-LDL and bind to UEA-1, and expressed the markers of endothelial cells, such as CD31, CD144 and vWF detected by immunocytochemistry. The transcription of CD31, KDR, CD144 and ENOS in both of them could be detected by RT-PCR, but FACS analysis showed significant differences of surface marker expression between them. In conclusion, two types of EPCs are successfully obtained by culturing MNCs isolated from human umbilical cord blood using endothelial cell-conditioned medium.


Subject(s)
Cell Separation , Cells, Cultured , Culture Media, Conditioned , Metabolism , Endothelial Cells , Cell Biology , Fetal Blood , Cell Biology , Humans , Leukocytes, Mononuclear , Cell Biology , Neovascularization, Physiologic , Physiology , Stem Cells , Cell Biology
17.
Article in Chinese | WPRIM | ID: wpr-267913

ABSTRACT

To investigate whether lymphocyte function-associated antigen 1 (LFA-1) and very late antigen 4 (VLA-4) are involved in vasoendothelial adhesion and transendothelial migration of high proliferative potential endothelial progenitor cells (HPP-EPCs), flow cytometry was used to analyze the expression of integrin beta1 and beta2, the expression of intercellular adhesion molecule (ICAM-1, 2) and vascular cell adhesion molecule (VCAM-1) in mouse bone marrow endothelial cells (mBMECs). The adhesion and transmigration through endothelial cells of the HPP-EPCs blocked by functional grade neutralizing antibodies of VLA-4 and LFA-1 were studied in vitro. The results revealed that HPP-EPCs were positive for CD11a and CD49d in HPP-EPCs. The expression of ICAM-1and VCAM-1 of mBMECs increased after activated by IL-1beta and TNF-alpha. The results of adhesion in vitro revealed that the numbers of the adhered and migrated cells in the CD11a antibody group, in the CD49d antibody group and in the combinational antibody group were less than those in the isotype control antibody group. Furthermore, the number of adhered and migrated cells in the combinational antibody group was less than that in the CD11a or the CD49d antibody group (p < 0.05). It is concluded that both LFA-1 and VLA-4 are involved in vasoendothelial adhesion and transendothelial migration of HPP-EPCs.


Subject(s)
Animals , Antigens, CD , Metabolism , Bone Marrow Cells , Cell Biology , Cell Adhesion , Cell Adhesion Molecules , Metabolism , Cell Movement , Cells, Cultured , Endothelial Cells , Cell Biology , Humans , Integrin alpha4beta1 , Physiology , Intercellular Adhesion Molecule-1 , Metabolism , Lymphocyte Function-Associated Antigen-1 , Physiology , Mice , Stem Cells , Cell Biology , Vascular Cell Adhesion Molecule-1 , Metabolism
18.
Article in Chinese | WPRIM | ID: wpr-308060

ABSTRACT

<p><b>OBJECTIVE</b>To identify the mutations of the tyrosinase gene (TYR) and P gene in patients with oculocutaneous albinism (OCA).</p><p><b>METHODS</b>Polymerase chain reaction (PCR) and denaturing high performance liquid chromatography (DHPLC) were applied to detect the mutations in all exons of TYR gene and P gene. Then DNA sequencing and restriction endonuclease analysis were used to confirm the mutations detected by DHPLC. Novel mutations were screened in 100 unrelated persons with normal phenotypes to exclude the possibility of polymorphism.</p><p><b>RESULTS</b>Two mutations were detected in the P gene of the three patients and none in TYR gene. Heterozygous mutation of T450M in exon 13 of the P gene was detected in patient 1. Patient 2 had a heterozygous mutation of T450M in exon 13 and a heterozygous mutation of G775R in exon 23 of the P gene. Patient 3 had a heterozygous mutation of G775R as well. Restriction endonuclease analysis of the P gene exon 13 showed that the Oli I site had partly disappeared resulting from the heterozygous mutation T450M in patient 1 and patient 2, but not in 100 unrelated individuals. The heterozygous mutation T450M is a novel mutation.</p><p><b>CONCLUSION</b>Gene diagnosis of OCA can be carried out effectively by combining PCR, DHPLC, DNA sequencing and restriction endonuclease analysis.</p>


Subject(s)
Albinism, Oculocutaneous , Genetics , Base Sequence , Catechol Oxidase , Genetics , Child, Preschool , DNA Mutational Analysis , Exons , Genetics , Female , Hermanski-Pudlak Syndrome , Genetics , Humans , Monophenol Monooxygenase , Genetics , Mutation , Young Adult
19.
Article in Chinese | WPRIM | ID: wpr-814097

ABSTRACT

OBJECTIVE@#To observe the inductive efficiency of deriving hematopoietic colony-forming cells from murine embryonic stem (mES) cells co-cultured with bone marrow stromal cell-conditional medium (mBMEC-CM).@*METHODS@#After the day-4 embryoid bodies (4 dEBs) were derived from embryonic stem cell-D3 (ES-D3) cells, the cells of 4 dEBs were induced into hematopoietic colony-forming cells by co-culturing with mBMEC-CM. The numbers of 4 dEB-derived hematopoietic colonies (high proliferation potential-colony formation cells and burst forming unit-erythroid, HPP-CFC and BFU-E) were detected to explore the relation between the implanted 4 dEB-derived cell numbers and the colony numbers of BFU-E and HPP-CFC. The inducing effect of mBMEC-CM was observed according to the doses and days of induction.@*RESULTS@#The number of 4 dEB-derived cells within 1 x 10(7)-4 x 10(7)/L was positively related to the colony numbers of HPP-CFC and BFU-E (HPP-CFC, r=0.916,P< 0.05; BFU-E, r=0.927, P<0.05). The inducing doses of mBMEC-CM within 0-20% were positively related to the colony numbers of HPP-CFC and BFU-E (HPP-CFC, r=0.909, P<0.05; BFU-E, r=0.927, P<0.01). The colony numbers of HPP-CFC and BFU-E derived from the 4 dEB-derived cells were the highest after 3 days of induction, followed by those of 6 days and 9 days.@*CONCLUSION@#Bone marrow endothelial cell-conditional medium can promote the generation of HPP-CFC and BFU-E from murine embryonic stem cells.


Subject(s)
Animals , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cells, Cultured , Coculture Techniques , Culture Media, Conditioned , Embryonic Stem Cells , Cell Biology , Endothelial Cells , Cell Biology , Hematopoietic Stem Cells , Cell Biology , Mice , Stromal Cells , Cell Biology
20.
National Journal of Andrology ; (12): 431-435, 2008.
Article in Chinese | WPRIM | ID: wpr-319218

ABSTRACT

<p><b>OBJECTIVE</b>To compare the proteomic differences between normal and necrozoospermic human spermatozoa.</p><p><b>METHODS</b>We isolated proteins from 30 ejaculates of 10 healthy donors and 3 specimens of a necrozoospermia patient by two-dimensional gel electrophoresis (2-DE), analyzed their 2-DE maps and identified some differentially expressed proteins by mass spectrometry.</p><p><b>RESULTS</b>A total of (905 +/- 57) and (792 +/- 28) proteins were isolated from the spermatozoal maps of the normal and necrozoospermic men, respectively, and 178 found to be differentially expressed in that of the necrozoospermia patient. The 6 missing protein spots in the necrozoospermic map were identified as sperm protein (human, accession No. 060904), zinc finger protein 174 (AW-1), F-actin capping protein alpha-3 subunit, testis-specific inhibitor of apoptosis, death domain receptor 3 soluble form (fragment) and peptide similar to the activator of CREM in the testis.</p><p><b>CONCLUSION</b>Six missing protein spots were identified in the necrozoospermic spermatozoal map which may be associated with the development of necrozoospermia.</p>


Subject(s)
Electrophoresis, Gel, Two-Dimensional , Humans , Male , Mass Spectrometry , Oligospermia , Metabolism , Pathology , Peptide Mapping , Proteome , Proteomics , Methods , Spermatozoa , Metabolism , Pathology
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